Concentration-dependent effects of rapamycin on proliferation, migration and apoptosis of endothelial cells in human venous malformation

Abstract

Rapamycin has been reported to be immunosuppressive and anti-proliferative towards vascular endothelial and smooth muscle cells. The purpose of the present study was to investigate the effects of rapamycin on the biological behaviors of endothelial cells that have been separated from the deformed vein in human venous malformation (VM). Cellular morphology was observed using inverted microscopy. An MTT assay was performed to measure the cell viability at different concentrations of rapamycin and different time points. Cell apoptosis and migration were detected using a terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling assay and a wound-healing assay, respectively. At 48 and 72 h, rapamycin inhibited proliferation of human VM endothelial cells, with the effects becoming more pronounced with increasing concentration. Only rapamycin at a concentration of 1,000 ng/ml had a significant effect at 24 h in repressing proliferation. At 48 h, compared with the blank group, the majority of cells maintained a clear nuclear boundary and a regular shape following treatment with 1 ng/ml rapamycin; 10 and 100 ng/ml rapamycin caused desquamation and rounded shape; and 1,000 ng/ml rapamycin caused even more marked desquamation, rounded shape and necrosis. Rapamycin at concentrations of 1, 10, 100 and 1,000 ng/ml reduced cell viability, increased the number of apoptotic cells and suppressed the migration capacity of human VM endothelial cells, and the effects became more pronounced with increasing concentration, when compared with the blank group. These findings provide evidence that rapamycin induces apoptosis and inhibits proliferation and migration of human VM endothelial cells in a concentration-dependent manner.

Keywords: cell viability; concentration-dependence; endothelial cells; rapamycin; venous malformation.

Figure 1.

Cellular morphology of human venous malformation endothelial cells. (A) Observation of morphology of human venous malformation endothelial cells (original magnification, ×200). (B) determination of the endovascular epithelial markers CD31, vWF, and CD31/vWF. CD, cluster of differentiation; vWF, von Willebrand factor; FITC, fluorescein isothiocyanate.

Figure 2.

Comparison of the effects of rapamycin on the cell viability of human venous malformation endothelial cells at different concentrations and different time points. Comparisons among multiple groups were performed using two-way analysis of variance followed by a Tukey's post hoc test. *P<0.05 vs. blank; ^#P<0.05 vs. 1 ng/ml rapamycin; ^&P<0.05 vs. 10 ng/ml rapamycin; ^$P<0.05 vs. 100 ng/ml rapamycin.

Figure 3.

Comparison of the effects of rapamycin on cellular morphology of human venous malformation endothelial cells at different concentrations. Cellular morphology of human venous malformation endothelial cells treatment with increasing concentrations of rapamycin (original magnification, ×400).

Figure 4.

Comparison of the effects of rapamycin on cell apoptosis of human venous malformation endothelial cells at different concentrations. (A) Terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling staining to evaluate cell apoptosis (original magnification, ×400). (B) The apoptosis rate of cells among different groups. Comparisons among multiple groups were performed using one-way analysis of variance followed by a Tukey's post hoc test. *P<0.05 vs. blank; ^#P<0.05 vs. 1 ng/ml rapamycin; ^&P<0.05 vs. 10 ng/ml rapamycin; ^$P<0.05 vs. 100 ng/ml rapamycin.

Figure 5.

Comparison of the effects of rapamycin on cell migration of human venous malformation endothelial cells at different concentrations. (A) The migration ability of cells detected by wound-healing assay (original magnification, ×200). (B) Migration rate of cells among different groups. Comparisons among multiple groups were performed using one-way analysis of variance followed by a Tukey's post hoc test. *P<0.05 vs. blank; ^#P<0.05 vs. 1 ng/ml rapamycin; ^&P<0.05 vs. 10 ng/ml rapamycin; ^$P<0.05 vs. 100 ng/ml rapamycin.